The ribosome catalyzes messenger RNA-directed protein synthesis by a mechanism common to all organisms that remains obscure despite decades of study. It is important that we understand how ribosomes perform their function for many reasons: (1) protein synthesis is a critically important synthetic process in all organisms, (2) the mechanism may involve novel enzymology due to the intimate involvement of RNA in ribosome function, and (3) knowledge of the mechanism is likely to have clinical impact because bacterial ribosomes are the targets of many antibiotics. Since lack of high-resolution information about the conformation of the ribosome and the macromolecules that interact with it is seriously limiting the process of this field, the focus of the work planned for the next four years is the determination of the structure of the ribosome, and of some of its macromolecular ligands by X-ray crystallography. Three projects will be undertaken. First, the crystal structure of the large ribosomal subunit from Haloarcula marismortui will be solved. Crystals are available that diffract to better than 4 A, and a first heavy atom derivative is already be in hand. A the same time, biochemical studies of the factors that limit the quality of these crystals and of ribosome crystals generally will continue, as will crystallization trials of complexes of elongation factor G (EF-G) with ribosomes. Crystallization trials will also be run with 70S ribosomes from several bacterial species. Second, domains of 30S ribosomal subunits will be prepared by reconstitution, and experiments done to find out if they can be crystallized. The crystallization of complexes of EF-G and RNA oligonucleotides derived from the sarcin/ricin and thiostrepton regions will also be explored, as will the crystallization of complexes of ribosomal protein L25 and oligonucleotides derived from 5S rRNA. The structures of all new crystals obtained that are suitable for crystallographic analysis will be pursued. Third, the crustal structure of elongation factor G (EF-G) complexed with a GTP analogue will be solved.